Methods For mRNA Poly(A) Sizing Using RNase Digestion, Capillary Gel Electrophoresis, And Nanopore-Based Sequencing

By Lauren Hinkel, Ryan Williams, Calleigh Herren, Theresa Legan, Vernal Biosciences

The poly(A) tail region of an mRNA functions to stabilize the mRNA and enable translation of the protein. Due to its biological importance of an mRNA’s poly(A) tail, the length of the poly(A) tail is typically evaluated as a Critical Quality Attribute for manufactured mRNA medicines. The method described here was developed to evaluate the length of mRNA poly(A) tails via a low-cost, high-throughput three-step method.

The mRNA samples for this method are prepared using RNase enzymes that digest the phosphodiester bonds that are 3’ to any base that is not an adenosine, leaving intact only the poly(A) tails. The samples are then purified to remove the RNases, individual nucleotides, and short runs of adenosines that are not part of the poly(A) tail. The poly(A) tail fragments are analyzed using fluorescence-based capillary gel electrophoresis (CGE) with a ssRNA poly(A) reference ladder to determine the size of the poly(A) fragments in the sample. The CGE results of the poly(A) fragments are confirmed using direct sequencing of the intact mRNA by the Oxford Nanopore Technologies (ONT) minION.